Terminal differentiation of macrophage-lineage cells is a critical stage in determining the functional attributes of mature macrophages. Because this process occurs after the circulating monocytes enter the tissues, it is difficult study, and relatively little is known about the regulation of this important transition. The applicant for this Clinical Investigator Development Award is a pediatric hematologist 3 years out of fellowship at Memorial Sloan-Kettering Cancer Center, with a research focus on the regulation of macrophage differentiation. The applicant's long-term goal is the development of effective macrophage-based immunotherapy regimens for the treatment of malignancy. The proposed project is an outgrowth of the applicant's ongoing studies comparing monocyte-derived macrophages differentiating in vitro under the control of macrophage colony- stimulating factor vs. those exposed to the pro-inflammatory cytokine interferon gamma. The project will examine a novel property which the applicant has recently identified, activation-induced macrophage apoptosis, which is regulated during macrophage differentiation by the interaction of MCSF and IFNgamma. Activation-induced apoptosis is rapid (6-12 hrs), complete (>90% of cells affected), and occurs only in those macrophages with a history of exposure to IFNgamma at some point during differentiation. Its importance may thus be as a physiologic check to limit uncontrolled activation of inflammatory macrophages. Preliminary data suggests that the regulatory enzyme protein kinase C (PKC) appears to play a central role in the signal-transduction pathway linking activation to cell death in the apoptosis-sensitive macrophages. However, both sensitive and resistant macrophage subtypes possess abundant PKC activity. The applicant's hypothesis is that differences must therefore exist either in the nature of the PKC found in the two types (e.g., due to expression of different isoenzymes), or in the downstream elements affected by activation of PKC (in particular the anti-apoptosis proto-oncogene BCL-2). The specific aims of this proposal are to compare the isoforms, subcellular localization, and response to activation of PKC in the apoptosis-sensitive and resistant macrophage subtypes, and to define the mechanism underlying the rapid decline in BCL-2 expression observed to occur following activation of the sensitive, but not the resistant, macrophage subtype. The applicant proposes to take advantage of the extensive expertise and facilities for the study of protein kinase C and signal transduction pathways available in the laboratory of Dr. Howard Rasmussen and colleagues at the applicant's institution. The applicant has been provided with a fully equipped laboratory by his department (Pediatrics) to pursue this project, and is being sponsored for this application by Dr John Hardin, Chairman of Medicine, and co-sponsored by Dr. Rasmussen. Completion of this project will offer insight into a previously undescribed aspect of macrophage biology, and will equip the applicant with sophisticated, state-of-the-art techniques for his long- term study of macrophage differentiation and activation.